Defects in mitochondria cause many human diseases and contribute to aging and age-related pathology. Mitochondria are organelles where oxidative phosphorylation (OXPHOS) occurs, the main energy- producing process in most cells. Accordingly, the pathological consequences of mitochondrial dysfunction are most apparent in tissues with high energy demands such as heart, brain and skeletal muscle. The downstream effects of mitochondrial dysfunction almost always involve disruption of OXPHOS and ATP production, but also can include oxidative stress due to increased reactive oxygen species (ROS), defective signal transduction, and cell death, that alone or together contribute to complex, tissue-specific disease pathology. Central to mitochondrial function is mitochondrial DNA (mtDNA), which is housed within the organelle and encodes thirteen essential proteins of the OXPHOS complexes. Thus, a primary mechanism controlling human mitochondrial gene expression and OXPHOS activity involves nucleus-encoded regulatory factors such as mitochondrial RNA polymerase and transcription factors that are imported into the organelle to initiate transcription, translation, and assembly of the mtDNA-encoded OXPHOS subunits. The overall goal of this competing renewal proposal is understand how mitochondrial gene expression is regulated in mammalian cells and how defects in this critical process contribute to heart disease. We will accomplish this goal through completion of three specific aims that focus on the mechanism of action of key mitochondrial transcriptional regulatory factors and their molecular interactions discovered in my laboratory during the current funding period. Specific Aim 1 is to determine how the dual-function h-mtTFB1 and h-mtTFB2 transcription factors/rRNA methyltransferases individually and cooperatively activate mitochondrial gene expression and biogenesis, and the mechanism underlying their coordinate regulation. Specific Aim 2 is to generate inducible, heart-specific knock-outs of mtTFB1 and mtTFB2 in mice to determine their roles in mitochondrial gene expression and normal heart function in vivo, and to model the mitochondrial pathology underlying cardiomyopathy. Specific Aim 3 is to determine the mechanism of activation of mitochondrial transcription by mitochondrial ribosomal protein L12 (which we discovered is a novel component of mitochondrial transcription complexes) and its functional consequences in vivo. The information gained from these studies will provide important new insight into the pivotal role of mitochondria and mtDNA in human diseases such as heart disease and a platform for development of therapeutic strategies that augment the function of these critical energy-producing organelles in cells. Mitochondria are membrane-bound compartments in cells that produce the vast majority of energy needed for major organs such as heart and brain to function. Mitochondria have their own DNA (called mtDNA) and defects in mitochondria or mtDNA cause many human diseases and contribute to aging and age-related pathology. This proposal is focused on understanding how mtDNA is regulated in human cells and will generate mouse models for how mitochondrial deficiencies causes heart disease.